Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen which is primarily a nosocomial infection, and is particularly serious for patients hospitalized with burns, cancer, and cystic fibrosis where the fatality rate is ~50%. P. aeruginosa infection is the fourth most common in U.S. hospitals with an incidence of ~10% of all nosocomial infections, and is associated with a low susceptibility to antibiotic treatment largely due to multidrug efflux pumps associated with antibiotic resistance genes.

Given the severity and incidence of P. aeruginosa infection, new means of treatment that are not subject to antibiotic efflux would be valuable additions to current therapy. This application describes studies to explore a new and innovative direction for the development of antimicrobials by exploiting the vulnerability represented by bacterial iron homeostasis when pathogens are confronted with the host immune system. Our studies are based on unique results demonstrating that (1) the function of bacterioferritin (BfrB) is required for P. aeruginosa growth in cystic fibrosis sputum, (2) the function of BfrB in bacterial iron homeostasis requires binding and electron delivery by a ferredoxin Bfd, (3) the crucial BfrB-Bfd interface, recently defined by our X-ray crystallographic work, is highly complementary, and (4) small molecule probes (fragments) interact with BfrB at the BfrB-Bfd interface.

Consequently, we propose to develop probes that by inhibiting the BfrB-Bfd association inhibit bacterial iron homeostasis and impair P. aeruginosa growth. We have shown that fragment based drug discovery (FBDD) is a viable way to identify small molecules that block the BfrB-Bfd interface and inhibit the release of iron from BfrB. Our objective will be achieved by integrating the following activities in an iterative fashion:

Utilize medicinal chemistry to drive iterative rounds of structure activity relationship development by parallel synthesis utilizing the following criteria: (i) in vitro binding to BfrB, (ii) inhibition of the interaction of BfrB with Bfd, (iii) inhibition of the release of iron from BfrB, (iv) inhibition of P. aeruginosa in a zone-killing assay.

The work has the potential to produce the first-ever chemical probes of the critical BfrB-Bfd interface, which are expected to provide critical insight as to the in vitro and in vivo function of BfrB, a promising target for the treatment of P. aeruginosa infection. Our long-term goal is to convert from a target validation to a drug discovery program, to discover and develop new therapeutics for the treatment of P. aeruginosa infection.

Dynamics and Interactions in the Release of Iron Stored in Bacterioferritin (2010-12)

The antibiotic resistance developed by bacteria is cause of public concern. A possible solution is to develop drugs that interfere with the handling of iron, a required nutrient. To approach this goal we plan to gain molecular understanding of the dynamic processes and inter-protein interactions that allow P. aeruginosa to store the toxic Fe2+ iron, and when needed, release it for its safe incorporation into metabolic paths that guarantee survival of the pathogen in a host.

Organisms that cause diseases such as respiratory tract infections (Haemophilus influenzae), enteric conditions (Shigella dysenteriae) and the opportunistic Pseudomonas aeruginosa have developed sophisticated mechanisms for sequestering iron from their host. This intense competition between invading pathogens and their host for the nutrient has led to the idea that new antimicrobials may target iron acquisition and homeostasis.

To approach more closely to this goal, it is important to gain molecular-level understanding of the mechanisms by which pathogens manage iron, from acquisition and internalization to storage and utilization. Significant advances have improved our understanding of iron uptake by P. aeruginosa and many other pathogens. In comparison, little is known about the fate of internalized iron. One mechanism whereby iron toxicity is controlled is by storage of iron in ferritin and bacterioferritin, which are large proteins capable of storing up to 4,000 iron atoms in their internal cavities. Despite the importance of ferritins and bacterioferritins in regulating iron concentrations and preventing its toxic effects, little is known about the processes that deliver Fe2+ for storage or the signals that prompt its release for safe integration in metabolism.

We have recently demonstrated that mobilization of Fe2+ from bacterioferritin A (BfrA) in P. aeruginosa requires electron transfer from a ferredoxin reductase (FPR). Thus the BfrA-FPR complex is an unprecedented opportunity to investigate how bacterioferritins recognize their physiological regulators and if binding modulates the dynamic properties of the bacterioferritin to facilitate iron release. To fill these gaps we plan to: (1) Investigate the dynamic properties of BfrA utilizing a strategy specifically tailored to study large proteins using hydrogen/deuterium H/D exchange coupled to NMR spectroscopy. (2) Investigate the dynamic properties of BfrA with the aid of computational methods and (3) Utilize computational and HD/NMR methods to investigate how BfrA binds to FPR and determine the effect that the inter-protein association exerts on the dynamic properties of BfrA.

Mechanisms of Heme Capture by the Hemophore Secreted by Pseudomonas aeruginosa (2008)

Heme is the most abundant source of circulating iron in mammals. It is therefore not surprising that many pathogenic bacteria, including the opportunistic Pseudomonas aeruginosa, avidly pursue its capture and internalization in order to overcome the very low free-iron concentrations encountered in their mammalian hosts. To capture heme, several pathogenic bacteria, including P. aeruginosa, deploy a heme acquisition system (Has), which consists of a protein secreted to the extracellular space (HasAp) and an outer membrane receptor (HasR). HasAp is also termed a hemophore because it efficiently captures hemoglobinheme and delivers it to the receptor for subsequent internalization.

The studies proposed herein aim to achieve fundamental molecular level understanding of the protein-protein interactions that allow HasAp to “steal” heme from human hemoglobin. In particular, the investigators seek to gain unprecedented structural, dynamic and mechanistic insights into the factors that determine the transfer of heme from human hemoglobin to HasAp.

This long-range goal will be reached by pursuing two main objectives:

Elucidate the three dimensional structure of apo-HasAp,

Identify the binding interface of the encounter complex that forms when HasAp binds to hemoglobin, prior to heme transfer, and decipher the role played by the gross reorganization of HasAp structural elements in the molecular recognition and binding to hemoglobin.